Characteristics and outcome of anti-hepatitis D virus positive patients with hepatocellular carcinoma.

BACKGROUND & AIMS: Chronic hepatitis D virus (HDV) often leads to end-stage liver disease and hepatocellular carcinoma (HCC). Comprehensive data pertaining to large populations with HDV and HCC are missing, therefore we sought to assess the characteristics, management, and outcome of these patients, comparing them to patients with hepatitis B virus (HBV) infection. METHODS: We analysed the Italian Liver Cancer database focusing on patients with positivity for HBV surface antigen and anti-HDV antibodies (HBV/HDV, n = 107) and patients with HBV infection alone (n = 588). Clinical and oncological characteristics, treatment, and survival were compared in the two groups. RESULTS: Patients with HBV/HDV had worse liver function [Model for End-stage Liver Disease score: 11 vs. 9, p < .0001; Child-Turcotte-Pugh score: 7 vs. 5, p < .0001] than patients with HBV. HCC was more frequently diagnosed during surveillance (72.9% vs. 52.4%, p = .0002), and the oncological stage was more frequently Milan-in (67.3% vs. 52.7%, p = .005) in patients with HBV/HDV. Liver transplantation was more frequently performed in HBV/HDV than in HBV patients (36.4% vs. 9.5%), while the opposite was observed for resection (8.4% vs. 20.1%, p < .0001), and in a competing risk analysis, HBV/HDV patients had a higher probability of receiving transplantation, independently of liver function and oncological stage. A trend towards longer survival was observed in patients with HBV/HDV (50.4 vs. 44.4 months, p = .106). CONCLUSIONS: In patients with HBV/HDV, HCC is diagnosed more frequently during surveillance, resulting in a less advanced cancer stage in patients with more deranged liver function than HBV alone. Patients with HBV/HDV have a heightened benefit from liver transplantation, positively influencing survival.

Cholestatic HCV Cryoglobulinemia: A New Clinical and Pathological Entity before and after Direct-Acting Antiviral Therapies-A Case-Control Study.

Twenty-nine patients with HCV infection (HCV+) and mixed cryoglobulinemia (MC+) were retrospectively selected and matched for age and sex with 31 HCV+ MC- patients. Biomarkers of cholestasis (direct bilirubin, alkaline phosphatase, and gamma-glutamyl transferase), HCV-RNA and genotype, and plasma cryoprecipitates were measured before and after virus eradication; liver histology and plasma cells (aggregation and distribution), observed blinded by two pathologists, were analyzed. Sixty participants (mean age: 56.5; range: 35-77, males: 50%) with HCV infection were enrolled. Cholestasis (≥2 pathologically increased cholestasis biomarkers) was significantly higher in the MC group (p = 0.02) and correlated with cryoglobulinemia (OR 6.52; p = 0.02). At liver histological assessment, plasma cells were significantly increased in the MC+ group (p = 0.004) and tended to form aggregates more than the control group (p = 0.05). At multivariate analysis with MC, age, HCV-RNA, HBV diabetes, and cirrhosis, cholestasis was only significantly correlated to MC (OR 8.30; p < 0.05). In 25% patients, MC persisted after virus eradication with new antiviral treatment. Our study identified for the first time an association between MC, cholestasis, and an increased number of intrahepatic plasma cells in chronic hepatitis C (CHC) patients before virus eradication. Future studies are required to understand how MC contributes to liver damage and how its persistence affects the patients' follow-up after antiviral therapies.

Bacterial infections as a risk factor for non-neoplastic portal vein thrombosis development in cirrhotic patients.

BACKGROUND: Portal vein thrombosis (PVT) and sepsis are common complications in patients with liver cirrhosis. Factors that lead to PVT are not completely understood. This study aimed to investigate the possible association between bacterial infections and the development of PVT in cirrhotic patients. PATIENTS AND METHODS: 202 consecutive cirrhotic patients without previous infections, followed at the Liver Unit in Verona Hospital, were enrolled from 2017 to 2021 (median follow-up 3.3 years). During the follow-up period, PVT was diagnosed by ultrasound, CT and/or MRI, and episodes of bacterial infections requiring hospitalization were recorded. Malignant PVT was an exclusion criterion. RESULTS: Of the 202 patients enrolled (68.3 % males, mean age 63.8 ± 11 years), 22 (10.8 %) developed PVT during the follow up. In patients with PVT, the prevalence of previous bacterial infections was significantly higher compared to patients without PVT (63.6% vs 31.1 %; p = 0.02). Cox regression analysis revealed that a history of bacterial infection was the only variable that demonstrated a significant association with the risk of de novo PVT occurrence (HR 4.04, 95 % CI: 1.68-9.65). CONCLUSION: in patients with liver cirrhosis bacterial infections are a predisposing factor for the following development of PVT. Further studies are needed to confirm this evidence.

Prevalence of portal vein thrombosis in non-alcoholic fatty liver disease: a meta-analysis of observational studies.

Portal vein thrombosis (PVT) is a common complication of cirrhosis as a result of portal hypertension and modification in the hemostatic balance. Accumulating evidence now suggests that patients with non-alcoholic fatty liver disease (NAFLD), especially those with advanced forms, have an increased risk of PVT. Hence, we performed a meta-analysis of observational studies to estimate the overall prevalence of PVT in patients with NAFLD and its advanced forms compared with patients with advanced liver diseases from other etiologies. We systematically searched PubMed, Scopus and Web of Science databases from the inception date to December 30th 2022, using predefined keywords, to identify observational studies. Meta-analysis was performed using random-effects modeling. We included five observational studies for a total of 225,571 patients. Of these, 26,840 (11.9%) patients had NAFLD, whereas the PVT prevalence was 8.5% (n = 2,280). When compared with patients with advanced liver diseases from other etiologies, patients with NAFLD and its advanced forms had a higher risk of prevalent PVT (OR 1.34, 100% CI 1.07-1.67 p < 0,01). The between-study heterogeneity was substantial (I2 = 88%). This meta-analysis suggests that compared with patients with advanced liver diseases from other etiologies, patient with NAFLD and its advanced forms had a higher risk of prevalent PVT. Further research is required to understand the complex link between NAFLD/NASH and PVT development.

Glomerular Hyperfiltration: A Marker of Fibrosis Severity in Metabolic Associated Steatotic Liver Disease in an Adult Population.

Glomerular hyperfiltration (GH) is an increase in the glomerular filtration rate, possibly progressing to chronic kidney disease (CKD). Metabolic-associated steatotic liver disease (MASLD) is linked to an increased risk of CKD, especially if fibrosis is present; however, the association between GH and MASLD has not been explored. To evaluate GH prevalence in MASLD and its possible correlation with liver fibrosis. 772 consecutive patients with ultrasound MASLD (mean age 47.3 ± 8.9 years, 67.1% males) were enrolled. GH was defined as estimated glomerular filtration rate (eGFR) greater than the upper quartile of values in the cohort. Liver stiffness measurement (LSM) by FibroScan ≥ 7.2 kPa suggested liver fibrosis. GH was present in 20% of patients, liver fibrosis in 30%. In total, 53.4% of the cohort was obese, 40.9% hypertensive, 36.3% diabetic and 70.8% dyslipidaemic. GH patients compared to non-GH were significantly younger (38.4 ± 8.3 vs. 49.5 ± 7.7, p < 0.001), with higher prevalence of LSM > 7.2 kPa (35.5% vs. 29%, p < 0.001), without any difference in metabolic comorbidities. In multivariate analysis, age (OR 0.85, CI 95% 0.82-0.87) and significant fibrosis (OR 1.83; CI 95%1.10-3.03) remained independently associated with GH, regardless of the presence of metabolic alterations and nephrotoxic drugs. GH, an early marker of renal damage, is highly prevalent in MASLD and is associated with hepatic fibrosis. GH may be considered an early marker of both liver and renal disease and its recognition could prompt the management of risk factors aimed at preventing the progression of both hepatic and renal disease.

Diagnostic accuracy of AGILE 3+ score for advanced fibrosis in patients with NAFLD: A systematic review and meta-analysis.

BACKGROUND AND AIMS: A simple noninvasive score, the Agile 3+ score, combining liver stiffness measurement, aspartate aminotransferase/alanine aminotransferase ratio, platelet count, diabetes status, sex, and age, has been proposed for the identification of advanced fibrosis in patients with suspected NAFLD. We performed a systematic review and meta-analysis of observational studies to evaluate the diagnostic accuracy of the Agile 3+ score in identifying patients with NAFLD and advanced fibrosis. Recently, an International consensus changed the nomenclature of NAFLD into metabolic-associated steatotic liver disease, so currently, the two terms are interchangeable. APPROACH AND RESULTS: We systematically searched MEDLINE, Ovid Embase, Scopus, and Cochrane Library electronic databases for full-text published articles in any language from the inception to the April 24, 2023. We included original articles reporting data on the sensitivity and specificity of the Agile 3+ score, according to previously described rule-out (≤ 0.451) and rule-in (≥ 0.679) cutoffs. We included 6 observational studies (total of 6955 participants) with biopsy-proven NAFLD [mean age 53 (SE 4) years, mean body mass index 30.9 (SE 2.3) kg/m 2 , 54.0% men, prevalence of diabetes 59.6%]. The pooled prevalence of advanced fibrosis (≥ F3) was 42.1%. By the rule-out cutoff, the overall sensitivity and specificity were 88% (95% CI: 81-93%; I2 = 89.2%) and 65% (95% CI: 54-75%; I2 = 97.6%), respectively. By the rule-in cutoff, the overall sensitivity and specificity were 68% (95% CI: 57-78%; I2 =91.1%) and 87% (95% CI: 80%-92%; I2 =96.7%), respectively. Meta-regression analyses reported that the diagnostic accuracy was partly mediated by age ( p < 0.01), body mass index ( p < 0.01), and, although not statistically significant, sex ( p = 0.06). CONCLUSIONS: Our systematic review and meta-analysis suggests that Agile 3+ accurately diagnoses NAFLD with advanced fibrosis and can identify patients eligible for biopsy and emerging pharmacotherapies.

Metabolic-associated fatty liver disease and liver fibrosis scores as COVID-19 outcome predictors: a machine-learning application.

Patients with COVID-19 and metabolic-dysfunction associated fatty liver disease (MAFLD) appear to be at higher risk for severe manifestations, especially in the youngest decades. Our aim was to examine whether patients with MAFLD and/or with increased liver fibrosis scores (FIB-4) are at risk for severe COVID-19 illness, using a machine learning (ML) model. Six hundred and seventy two patients were enrolled for SARS-CoV-2 pneumonia between February 2020 and May 2021. Steatosis was detected by ultrasound or computed tomography (CT). ML model valuated the risks of both in-hospital death and prolonged hospitalizations (> 28 days), considering MAFLD, blood hepatic profile (HP), and FIB-4 score. 49.6% had MAFLD. The accuracy in predicting in-hospital death was 0.709 for the HP alone and 0.721 for HP + FIB-4; in the 55-75 age subgroup, 0.842/0.855; in the MAFLD subgroup, 0.739/ 0.772; in the MAFLD 55-75 years, 0.825/0.833. Similar results were obtained when considering the accuracy in predicting prolonged hospitalization. In our cohort of COVID-19 patients, the presence of a worse HP and a higher FIB-4 correlated with a higher risk of death and prolonged hospitalization, regardless of the presence of MAFLD. These findings could improve the clinical risk stratification of patients diagnosed with SARS-CoV-2 pneumonia.

Interleukin-6: A New Marker of Advanced-Sarcopenic HCC Cirrhotic Patients.

Hepatocellular carcinoma (HCC) is the major cause of liver-related death worldwide. Interleukin 6 (IL-6) promotes the growth of the HCC microenvironment. The correlation between Child-Pugh (CP) and HCC stage and between HCC stage and sarcopenia is still not clear. Our aim was to investigate whether IL-6 is correlated with HCC stage and could represent a diagnostic marker for sarcopenia. Ninety-three HCC cirrhotic patients in different stages, according to BCLC-2022 (stages A, B, and C), were enrolled. Anthropometric and biochemical parameters, comprehensive of IL-6, were collected. The skeletal muscle index (SMI) was measured using dedicated software on computer tomography (CT) images. IL-6 level was higher in advanced (BCLC C) compared to the early-intermediate (BCLC A-B) stages (21.4 vs. 7.7 pg/mL, p < 0.005). On multivariate analysis, IL-6 levels were statistically dependent on the degree of liver disease severity (CP score) and HCC stages (p = 0.001 and p = 0.044, respectively). Sarcopenic patients presented lower BMI (24.7 ± 5.3 vs. 28.5 ± 7.0), higher PMN/lymphocyte ratio (2.9 ± 2.4 vs. 2.3 ± 1.2) and increased values of log (IL-6) (1.3 ± 0.6 vs. 1.1 ± 0.3). Univariate logistic regression between sarcopenia and log (IL-6) showed a significant odds ratio (OR 14.88, p = 0.044) with an AUC of 0.72. IL-6 appears to be an effective biomarker for the diagnosis of advanced cirrhotic HCC. In addition, IL-6 could be considered a marker of cirrhotic HCC-related sarcopenia, suggesting further investigation with BIA- or CT-dedicated software.

SerpinB3 as hepatic marker of post-resective shear stress.

Post-resective liver failure is a frequent complication of liver surgery and it is due to portal hyperperfusion of the remnant liver and to arterial vasoconstriction, as buffer response of the hepatic artery. In this context, splenectomy allows a reduction of portal flow and increases the survival chance in preclinical models. SerpinB3 is over-expressed in the liver in oxidative stress conditions, as a mechanism of cell defense to provide survival by apoptosis inhibition and cell proliferation. In this study, the expression of SerpinB3 was assessed as predictor of liver damage in in vivo models of major hepatic resection with or without splenectomy. Wistar male rats were divided into 4 groups: group A received 30% hepatic resection, group B > 60% resection, group C > 60% resection with splenectomy and group D sham-operated. Before and after surgery liver function tests, echo Doppler ultrasound and gene expression were assessed. Transaminase values and ammonium were significantly higher in groups that underwent major hepatic resection. Echo Doppler ultrasound showed the highest portal flow and resistance of the hepatic artery in the group with > 60% hepatectomy without splenectomy, while the association of splenectomy determined no increase in portal flow and hepatic artery resistance. Only the group of rats without splenectomy showed higher shear-stress conditions, reflected by higher levels of HO-1, Nox1 and of Serpinb3, the latter associated with an increase of IL-6. In conclusion, splenectomy controls inflammation and oxidative damage, preventing the expression of Serpinb3. Therefore, SerpinB3 can be considered as a marker of post-resective shear stress.

Spur cells in liver cirrhosis are predictive of acute-on-chronic liver failure and liver-related mortality regardless of severe anaemia.

Chronic anaemia in advanced liver disease is a frequent finding. The aim was to explore the clinical impact of spur cell anaemia, a rare entity typically associated with end-stage of the disease. One-hundred and nineteen patients (73.9% males) with liver cirrhosis of any etiology were included. Patients with bone marrow diseases, nutrients deficiencies and hepatocellular carcinoma were excluded. In all patients, a blood sample was collected to check for the presence of spur cells on blood smear. A complete blood biochemical panel was recorded together with Child-Pugh (CP) score and Model for End-Stage Liver Disease (MELD) score. For each patients, clinically relevant events, such as acute-on-chronic liver failure (ACLF) and 1 year liver-related mortality, were registered. Patients were then grouped according to the percentage of spur cells at smear (> 5%, 1-5%, < 1%). Severe anaemia was defined as haemoglobin levels lower than 8 g/dL. 9.2% of subjects had > 5% spur cells, only 2 had evidence of haemolysis. In patients with > 5% spur cells, haemoglobin and albumin were lower compared with the other sub-group, while MELD score, CP score, International Normalized Ratio, ferritin, creatinine and unconjugated bilirubin were higher. Patients with more spur cells were more decompensated and developed more frequently ACLF. ACLF and liver-related mortality were significantly and independently associated with the presence of > 5% spur cells but not with baseline severe anaemia. Cirrhotic patients have a fairly high prevalence of spur cells, not always associated with severe haemolytic anaemia. The presence of spur red cells is per se associated with a worse prognosis and, therefore, should be always evaluated to prioritize patients for intensive management and eventually liver transplantation.

Chronic anaemia is a frequent finding in liver cirrhosis and spur cell anaemia has been shown to be an uncommon non-immune haemolytic disease typically related to advanced-liver disease. In our study, spur cell anaemia (spur cells >5%) was found in almost 10% of outpatient cirrhotics and was significantly related to more decompensated disease, higher incidence of ACLF and 1 year liver-related mortality. More importantly, the vast majority of patients with high percentage of spur cells did not have severe anaemia. Therefore, spur cells should be searched for in patients with advanced liver disease by a simple blood smear evaluation, even in the absence of significant anaemia, because of relevant prognostic impact and in order to prioritize patients to intensive management and possibly liver transplantation.

The multi-drug resistant organisms infections decrease during the antimicrobial stewardship era in cirrhotic patients: An Italian cohort study.

BACKGROUND AND PURPOSE: Bacterial infections represent a major cause of morbidity and mortality in cirrhotic patients. Our aim was to assess the incidence of bacterial infections, in particular due to multidrug-resistant organisms (MDROs) before and after the introduction of the antimicrobial stewardship program, "Stewardship Antimicrobial in VErona" (SAVE). In addition, we also analysed the liver complications and the crude mortality during the whole follow up. METHODS: We analysed 229 cirrhotic subjects without previous hospitalization for infections enrolled at the University Verona Hospital from 2017 to 2019 and followed up until December 2021 (mean follow-up 42.7 months). RESULTS: 101 infections were recorded and 31.7% were recurrent. The most frequent were sepsis (24.7%), pneumonia (19.8%), spontaneous bacterial peritonitis (17.8%). 14.9% of infections were sustained by MDROs. Liver complications occurred more frequently in infected patients, and in case of MDROs infections with a significantly higher MELD and Child-Pugh score. In Cox regression analysis, mortality was associated with age, diabetes and bacterial infections episodes (OR 3.30, CI 95%: (1.63-6.70). Despite an increase in total infections over the past three years, a decrease in the incidence rate in MDROs infections was documented concurrently with the introduction of SAVE (IRD 28.6; 95% CI: 4.6-52.5, p = 0.02). CONCLUSIONS: Our study confirms the burden of bacterial infections in cirrhotic patients, especially MDROs, and the strong interconnection with liver complications. The introduction of SAVE decreased MDROs infections. Cirrhotic patients require a closer clinical surveillance to identify colonized patients and avoid the horizontal spread of MDROs in this setting.

Subclinical and clinical atherosclerosis in non-alcoholic fatty liver disease is associated with the presence of hypertension.

BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) is associated with increased cardiovascular (CV) risk. However, it is unclear whether NAFLD contributes independently to the development of CV disease. Our study aimed at assessing the differences in several indices of atherosclerosis, arterial stiffness and cardiac morphology among patients with isolated NAFLD, isolated hypertension (HT) or a combination of the two conditions. METHODS AND RESULTS: A total of 169 participants (mean age = 50.4 ± 10.2 yrs; males = 73.6%) were divided according to the presence of NAFLD and HT into three groups: only NAFLD (55 patients), only HT (49 patients), and NAFLD + HT (65 patients). Exclusion criteria were a BMI≥35 kg/m2 and a diagnosis of diabetes mellitus. Carotid ultrasonography was performed to measure markers of atherosclerosis and arterial stiffness. Cardiac remodeling was analyzed using echocardiography. The prevalence of subclinical and overt atherosclerosis was significantly higher in the NAFLD + HT patients as compared to the other two groups (atherosclerotic plaques: 43.1%, 10.9%, and 22.4% (p < 0.001) in NAFLD + HT, NAFLD, and HT groups, respectively). No differences were found among indices of arterial stiffening and cardiac remodeling across the three groups. In multivariate regression analysis, the coexistence of NAFLD and HT was an independent risk factor for overt atherosclerosis (OR = 4.88, CI 95% 1.14-20.93), while no association was found when either NAFLD or HT was considered alone. CONCLUSION: Overt atherosclerosis was significantly present only in NAFLD + HT patients, but not in patients with isolated NAFLD. This implies that the impact of NAFLD on vascular structure and function could depend on the coexistence of other major CV risk factors, such as HT.

Effects of direct-acting antiviral agents on lipid and glucose profile in HCV patients with type 2 diabetes: A real-life Italian experience.

OBJECTIVES: Hepatitis C virus (HCV) infection is associated with an increased risk of type 2 diabetes mellitus (T2DM) and cardiovascular diseases. The impact of HCV eradication on the metabolic profile in diabetic patients treated with direct-acting antiviral agents (DAAs) is not well defined. The aim of our study was to evaluate the effects of DAAs on a lipid and glucose profile in a cohort of diabetic patients with different liver fibrotic stages. METHODS: T2DM patients with active HCV infection were consecutively enrolled in this prospective trial. Glycolipidic status was assessed, before starting DAA treatment (T0) and at 12 months after the beginning of treatment (T1). Liver fibrotic stage was assessed by FibroScan. RESULTS: In all, 131 patients were enrolled and all of them achieved a sustained virologic response. At baseline, no significant differences were found in lipid and glucose profiles in subgroup analysis by liver fibrosis, HCV genotype, and cardiovascular risk factors. At T1, total cholesterol and low-density lipoprotein cholesterol, but not triglycerides, significantly increased irrespective of liver fibrotic stage and baseline anthropometric and clinical profiles, while glycated hemoglobin significantly decreased only in F4 patients. CONCLUSIONS: HCV eradication in diabetic patients is associated with a worsening lipid profile that could impact future cardiovascular risk. A careful global monitoring of cardiovascular risk factors in all diabetic patients after HCV eradication is needed.

Platelets in Non-alcoholic Fatty Liver Disease.

Non alcoholic steatohepatitis (NASH) is the inflammatory reaction of the liver to excessive accumulation of lipids in the hepatocytes. NASH can progress to cirrhosis and hepatocellular carcinoma (HCC). Fatty liver is the hepatic manifestation of metabolic syndrome. A subclinical inflammatory state is present in patients with metabolic alterations like insulin resistance, type-2 diabetes, obesity, hyperlipidemia, and hypertension. Platelets participate in immune cells recruitment and cytokines-induced liver damage. It is hypothesized that lipid toxicity cause accumulation of platelets in the liver, platelet adhesion and activation, which primes the immunoinflammatory reaction and activation of stellate cells. Recent data suggest that antiplatelet drugs may interrupt this cascade and prevent/improve NASH. They may also improve some metabolic alterations. The pathophysiology of inflammatory liver disease and the implication of platelets are discussed in details.

Comparison of Fondaparinux and Low-Molecular-Weight Heparin in the Treatment of Portal Vein Thrombosis in Cirrhosis.

BACKGROUND: Portal vein thrombosis is the most common thrombotic complication in cirrhosis. About 60% of anticoagulated patients can achieve recanalization. Despite fondaparinux (FPX) theoretical advantages, data are lacking about safety and efficacy for treatment of portal vein thrombosis in cirrhosis. METHODS: Cirrhotic patients with portal vein thrombosis treated with FPX or low-molecular-weight heparin (LMWH) were retrospectively included. The extension of thrombosis at baseline and its evolution during anticoagulant treatment were evaluated. Patients were treated with LMWH or FPX at therapeutic dosage and reduction was considered in selected cases. RESULTS: There were 124 patients included. Main portal vein branch, splenic, and superior mesenteric veins were involved in 84%, 13%, and 36% of cases, respectively. Forty-one patients (33%) were treated with FPX and 83 (67%) with LMWH. The probability of resolution of thrombosis at 36 months was significantly higher in patients treated with FPX than in those treated with LMWH (77% vs 51%; P = .001), particularly when prescribed at reduced dose. With multivariate analysis, the treatment with FPX (hazard ratio 2.38; P = .002) and use of a full dose (hazard ratio 1.78; P = .035) were independent predictors of portal vein full recanalization. Bleeding rate was higher in patients treated with FPX than in those treated with LMWH (27% vs 13%; P = .06). CONCLUSIONS: FPX appears to be more effective than LMWH in the treatment of portal vein thrombosis when used at reduced dose, also in complete thrombosis. FPX should be considered among possible treatments for portal vein thrombosis in cirrhosis.

Hyperdynamic circulatory syndrome in a mouse model transgenic for SerpinB3.

INTRODUCTION AND OBJECTIVES: SerpinB3 is a cysteine protease inhibitor involved in several biological activities. It is progressively expressed in chronic liver disease, but not in normal liver. The role in vascular reactivity of this serpin, belonging to the same family of Angiotensin II, is still unknown. Our aim was to evaluate the in vivo and in vitro effects of SerpinB3 on systemic and splanchnic hemodynamics. MATERIAL AND METHODS: Different hemodynamic parameters were evaluated by ultrasonography in two colonies of mice (transgenic for human SerpinB3 and C57BL/6J controls) at baseline and after chronic carbon tetrachloride (CCl4) treatment. In vitro SerpinB3 effect on mesenteric microvessels of 5 Wistar-Kyoto rats was analyzed measuring its direct action on: (a) preconstricted arteries, (b) dose-response curves to phenylephrine, before and after inhibition of angiotensin II type 1 receptors with irbesartan. Hearts of SerpinB3 transgenic mice and of the corresponding controls were also analyzed by morphometric assessment. RESULTS: In SerpinB3 transgenic mice, cardiac output (51.6±21.5 vs 30.1±10.8ml/min, p=0.003), hepatic artery pulsatility index (0.85±0.13 vs 0.65±0.11, p<0.001) and portal vein blood flow (5.3±3.2 vs 3.1±1.8ml/min, p=0.03) were significantly increased, compared to controls. In vitro, recombinant SerpinB3 had no direct hemodynamic effect on mesenteric arteries, but it increased their sensitivity to phenylephrine-mediated vasoconstriction (p<0.01). This effect was suppressed by inhibiting angiotensin II type-1 receptors. CONCLUSIONS: In transgenic mice, SerpinB3 is associated with a hyperdynamic circulatory syndrome-like pattern, possibly mediated by angiotensin receptors.

Myocardial overexpression of ANKRD1 causes sinus venosus defects and progressive diastolic dysfunction.

AIMS: Increased Ankyrin Repeat Domain 1 (ANKRD1) levels linked to gain of function mutations have been associated to total anomalous pulmonary venous return and adult cardiomyopathy occurrence in humans. The link between increased ANKRD1 level and cardiac structural and functional disease is not understood. To get insight into this problem, we have generated a gain of function ANKRD1 mouse model by overexpressing ANKRD1 in the myocardium. METHODS AND RESULTS: Ankrd1 is expressed non-homogeneously in the embryonic myocardium, with a dynamic nucleo-sarcomeric localization in developing cardiomyocytes. ANKRD1 transgenic mice present sinus venosus defect, which originates during development by impaired remodelling of early embryonic heart. Adult transgenic hearts develop diastolic dysfunction with preserved ejection fraction, which progressively evolves into heart failure, as shown histologically and haemodynamically. Transgenic cardiomyocyte structure, sarcomeric assembly, and stability are progressively impaired from embryonic to adult life. Postnatal transgenic myofibrils also present characteristic functional alterations: impaired compliance at neonatal stage and impaired lusitropism in adult hearts. Altogether, our combined analyses suggest that impaired embryonic remodelling and adult heart dysfunction in ANKRD1 transgenic mice present a common ground of initial cardiomyocyte defects, which are exacerbated postnatally. Molecular analysis showed transient activation of GATA4-Nkx2.5 transcription in early transgenic embryos and subsequent dynamic transcriptional modulation within titin gene. CONCLUSIONS: ANKRD1 is a fine mediator of cardiomyocyte response to haemodynamic load in the developing and adult heart. Increased ANKRD1 levels are sufficient to initiate an altered cellular phenotype, which is progressively exacerbated into a pathological organ response by the high ventricular workload during postnatal life. Our study defines for the first time a unifying picture for ANKRD1 role in heart development and disease and provides the first mechanistic link between ANKRD1 overexpression and cardiac disease onset.

Development of NASH in Obese Mice is Confounded by Adipose Tissue Increase in Inflammatory NOV and Oxidative Stress.

AIM: Nonalcoholic steatohepatitis (NASH) is the consequence of insulin resistance, fatty acid accumulation, oxidative stress, and lipotoxicity. We hypothesize that an increase in the inflammatory adipokine NOV decreases antioxidant Heme Oxygenase 1 (HO-1) levels in adipose and hepatic tissue, resulting in the development of NASH in obese mice. METHODS: Mice were fed a high fat diet (HFD) and obese animals were administered an HO-1 inducer with or without an inhibitor of HO activity to examine levels of adipose-derived NOV and possible links between increased synthesis of inflammatory adipokines and hepatic pathology. RESULTS: NASH mice displayed decreased HO-1 levels and HO activity, increased levels of hepatic heme, NOV, MMP2, hepcidin, and increased NAS scores and hepatic fibrosis. Increased HO-1 levels are associated with a decrease in NOV, improved hepatic NAS score, ameliorated fibrosis, and increases in mitochondrial integrity and insulin receptor phosphorylation. Adipose tissue function is disrupted in obesity as evidenced by an increase in proinflammatory molecules such as NOV and a decrease in adiponectin. Importantly, increased HO-1 levels are associated with a decrease of NOV, increased adiponectin levels, and increased levels of thermogenic and mitochondrial signaling associated genes in adipose tissue. CONCLUSIONS: These results suggest that the metabolic abnormalities in NASH are driven by decreased levels of hepatic HO-1 that is associated with an increase in the adipose-derived proinflammatory adipokine NOV in our obese mouse model of NASH. Concurrently, induction of HO-1 provides protection against insulin resistance as seen by increased insulin receptor phosphorylation. Pharmacological increases in HO-1 associated with decreases in NOV may offer a potential therapeutic approach in preventing fibrosis, mitochondrial dysfunction, and the development of NASH.

Heterotopic Implantation of Decellularized Pulmonary Artery Homografts In A Rodent Model: Technique Description and Preliminary Report.

PURPOSE: Despite a substantial amount of literature on tissue-guided regeneration, decellularization process, repopulation time points and stem cell turnover, more in-depth study on the argument is required. Currently, there are plenty of reports involving large animals, as well as clinical studies facing cardiac repair with decellularized homografts, but no exhaustive rodent models are described. The purpose of this study was to develop such a model in rats; preliminary results are also herein reported. MATERIAL AND METHODS: Fresh or decellularized pulmonary homografts from wild type rats were implanted in the abdominal aorta of green fluorescent protein positive rats. Three experimental groups were build up: sham, fresh homograft recipients and decellularized homograft recipients. The homograft decellularization process was performed with three cycles of detergent-enzymatic treatment protocol. Surgical technique of pulmonary homograft implantation and postoperative ultrasonographic evaluation were also reported; gross, histology and immunohistochemistry analysis on unimplanted and postoperative homografts were also carried out. RESULTS: The median total recipient operating time was 148 minutes, with a surgical success rate of 82%. The decellularization protocol resulted effective and showed a complete decellularization with intact extracellular matrix. At 15 days from surgery, the implanted decellularized pulmonary homografts exhibited cell repopulation in the outer media wall and partial endothelial lining in absence of rejection. CONCLUSIONS: Our technique is a feasible and reproducible model that can be fundamental for building a valid study for further exploitation on the field. Even in a short-term follow up, the decellularized pulmonary homografts showed autologous repopulation in absence of rejection.